Inhibitors of the urokinase receptor

ABSTRACT

The present invention concerns peptides as inhibitors of the binding of urokinase to the urokinase receptor. The peptides, which are preferably cyclic, are suitable as pharmaceutical agents for diseases that are mediated by urokinase and its receptor.

[0001] The present invention concerns peptides as inhibitors of thebinding of urokinase to the urokinase receptor. These peptides which arepreferably cyclic are suitable as pharmaceutical agents for diseaseswhich are mediated by urokinase and its receptor.

[0002] The serine protease uPA (urokinase-type plasminogen activator) isresponsible for various physiological and pathological processes such asthe proteolytic degradation of extracellular matrix material which isnecessary for the invasiveness and migration of cells and for tissueremodelling. uPA binds with high affinity (K_(D)=10⁻¹⁰−10⁻⁹M) to themembrane-based uPA receptor (uPAR) on the cell surface.

[0003] The binding of uPA to its receptor is involved in many invasivebiological processes such as the metastatic spread of malignant tumours,trophoplast implantation, inflammation and angiogenesis. Henceantagonists of uPA are able to inhibit the invasiveness, metastaticspread and angiogenesis of tumours. uPA antagonists can be used asagents for the treatment of invasive and metastasising cancer diseasesin which uPA and UPAR occur at the invasive foci of tumours (Dano etal., The receptor for urokinase plasminogen activator: Stromal cellinvolvement in extracellular proteolysis during cancer invasion, in:Proteolysis and Protein Turnover, Barrett, A. J. and Bond, J., Editor,Portland Press, London, 1994, 239) e.g. in cancers of the breast, lung,intestine and ovaries. In addition uPA antagonists can also be used forother purposes in which it is necessary to inhibit the proteolyticactivation of plasminogen, for example to treat diseases such asarthritis, inflammation, osteoporosis, retinopathies and forcontraception.

[0004] The uPA receptor is described in WO 90/12091 and in thepublications by Ploug et al., J. Biol. Chem. 268 (1993), 17539 and Ronneet al., J. Immunol. Methods 167 (1994), 91.

[0005] uPA is synthesized as a single chain molecule (pro-uPA) and isconverted enzymatically into an active two-chain uPA. The uPA moleculeis composed of three structurally independent domains, the N-terminalgrowth factor-like domain (GFD, uPA 1-46), a kringle structure domain(uPA 45-135) and the serine protease domain (uPA 159-411). GFD and thekringle domain together form the so-called aminoterminal fragment of uPA(ATF, uPA 1-135) which is produced by further proteolytic cleavage oftwo-chain uPA. ATF binds to the uPA receptor with a similar affinity asuPA.

[0006] The receptor-binding region of uPA spans the region of the aminoacids 12 to 32 since a peptide which contains the amino acid residues 12to 32 of uPA (in which case cysteine is replaced by alanine in position19) competes with ATF for binding to the uPA receptor (Appella et al.,J. Biol. Chem. 262 (1987), 4437-4440). In this publication it was alsoshown that this peptide also has an affinity for the uPA receptor aftercyclization by bridging the two cysteine residues at positions 12 and32. In an alternative approach Goodson et al., (Proc. Natl. Acad. USA 91(1994), 7129-7133) identified antagonistic uPA peptides for the uPAR byscreening a bacteriophage peptide library. These peptides had noapparent sequence homology to the natural uPAR-binding sequence of uPA.

[0007] Further investigations of the uPAR-binding region of uPA aredescribed in recent publications (Rettenberger et al., Biol. Chem.Hoppe-Seyler 376 (1995), 587-594); Magdolen et al., Eur. J. Biochem. 237(1996), 743-751; Goretzki et al., Fibrinolysis and Proteolysis 11(1997), 11-19). The residues Cys19, Lys23, Tyr24, Phe25, Ile28, Trp30and Cys31 were identified as important determinants for a uPA/uPARinteraction. In these investigations a uPA peptide having the aminoacids 16 to 32 of uPA was identified as the most effective inhibitor.

[0008] Magdolen et al., (1996) supra analysed the UPAR binding region ofthe uPA molecule using a peptide having the amino acids 14 to 32 of uPAand peptides derived therefrom. However, these peptides and alsopeptides used by other research groups (cf. e.g. Appella et al., (1987)supra) have a relatively low affinity for UPAR.

[0009] WO-A-94/22646 discloses linear peptides with a length of 6 to 18amino acids which are derived from the region of the amino acids 14 to33 of uPA. It is described that short peptides derived from uPA (uPA21-29 and uPA 21-26) are able to influence the growth of keratinocytes.Although WO-A-94/22646 makes reference to a potential use of the claimedpeptides to block the uPA/uPAR interaction, no data or informationwhatsoever are shown on such binding studies. Moreover, the peptides uPA21-29 and uPA 21-26 which are said to be preferred linear peptides donot contain the minimal UPAR binding region of linear uPA peptides whichcomprises the sequence region of amino acids 19 to 31. Hence theinfluence of the growth of keratinocytes by these short peptides is veryprobably not due to a uPA/uPAR interaction.

[0010] However, a disadvantage of the previously known uPA peptideinhibitors is that their affinity of binding to the uPA receptor isrelatively low and inadequate for a therapeutic application. Thus thereis a great need for new uPA peptide antagonists which have a higheraffinity for the receptor.

[0011] In quantitative investigations it was surprisingly found that thelinear peptide uPA (19-31), cyclic derivatives of this peptide andsequence-modified peptides from this uPA region have a considerablyimproved affinity of binding to the uPA receptor.

[0012] Experimental data demonstrate that the peptides according to theinvention can be used as uPA antagonists which bind with high affinityto the uPAR. Cyclic peptides are particularly preferred which arecharacterized by bridges, especially disulfide bridges, which do notoccur in the native uPA molecule.

[0013] Hence the present invention concerns peptides having the generalstructural formula (I):

[0014] in which

[0015] X²¹ to X³⁰ each denotes an aminocarboxylic acid, preferably anα-aminocarboxylic acid and X²¹ and X²⁹ are bridged together,

[0016] Y is a spacer

[0017] m and n are each independently 0 or 1,

[0018] and the monomeric building blocks are linked by —NR¹CO— or—CONR¹— bonds where R¹ in each case independently denotes hydrogen,methyl or ethyl, and pharmaceutically compatible salts and derivativesthereof.

[0019] The monomeric building blocks X²¹ to X³⁰ have preferably thefollowing meanings:

[0020] X²¹ and X²⁹ are α-aminocarboxylic acid building blocks which canbe bridged together and they particularly preferably have an SH sidechain, in particular a cysteine side chain or a structurally relatedside chain e.g. a penicillamine side chain. Alternatively X²¹ and X²⁹can also be two α-aminocarboxylic acid residues linked by a thioethergroup e.g. a lanthionine group.

[0021] X²² and X²⁷ are each independently α-aminocarboxylic acids withan aliphatic side chain, preferably an aliphatic hydrophilic side chainand in particular an amide side chain such as asparagine or glutamine,in particular asparagine.

[0022] X²³ is an α-aminocarboxylic acid with a basic side chain e.g.lysine, ornithine or arginine or with an aliphatic hydrophilic sidechain e.g. with an amide side chain such as glutamine or asparagine. X²³is particularly preferably lysine.

[0023] X²⁴ to X²⁵ are each independently α-aminocarboxylic acids with anaromatic side chain such as tyrosine, phenylalanine or tryptophan. X²⁴is particularly preferably tyrosine and X²⁵ is phenylalanine.

[0024] X²⁶ is an α-aminocarboxylic acid with an aliphatic side chain,preferably with an aliphatic hydrophilic side chain such ashydroxyvaline, homoserine, serine or threonine, in particular serine.However, X²⁶ can also have an aliphatic hydrophobic side chain such asalanine.

[0025] X²⁸ is an α-aminocarboxylic acid with an aliphatic side chain,preferably with an aliphatic hydrophobic side chain such as valine,norvaline, norleucine, isoleucine, leucine or alanine. X²⁸ isparticularly preferably isoleucine.

[0026] X³⁰— if present—is an α-aminocarboxylic acid with an aromaticside chain, preferably with a tryptophan side chain. The tryptophan sidechain can be optionally modified for example by reduction.

[0027] The peptides according to the invention are preferably derivedfrom the uPA sequence and contain at least 2 and particularly preferablyat least 3, for example 4 amino acid residues which also occur atcorresponding positions in the native uPA sequence. At least two of theamino acid residues X²², X²³, X²⁴, X²⁵, X²⁶, X²⁸ and X³⁰ particularlypreferably have a side chain which is identical to an amino acid at thesame position in the native uPA sequence. Most preferably at least 2 ofthe amino acid residues X²⁴, X²⁵, X²⁸ and—if present—X³⁰ have the sameside chain as in the native uPA sequence.

[0028] Y is a spacer group e.g. a peptidic spacer group composed of oneor several amino acids e.g. poly-Lys or another spacer group e.g. apolyethylene glycol group. The peptide can be coupled to carriersubstances via the group Y.

[0029] Hence a further subject matter of the present invention arecyclic peptides with a nine-membered ring of which at least two,preferably at least 3 and particularly preferably at least 4 of theamino acids forming the ring have a sequence from the uPA region 22 to28.

[0030] In addition to peptides having the structural formula (I),pharmaceutically compatible salts and derivatives thereof are alsosuitable as uPA antagonists. Suitable derivatives are in particularcompounds in which the reactive groups of the side chain or/and of theN-terminus or C-terminus e.g. amino or carboxylic acid groups have beenmodified. Examples of such modifications are acylation e.g. anacetylation of amino groups or/and an amidation or esterification ofcarboxylic acid groups.

[0031] Natural amino acids or enantiomers thereof ornon-naturally-occurring amino acids such as γ-aminobutyric acid,β-alanine can be used as the aminocarboxylic acids that the buildingblocks for the peptides according to the invention.

[0032] The monomeric building blocks are linked by acid amide bondsNR¹CO or CONR¹ i.e. the direction of the peptide sequence can also bereversed (retropeptides). As in native polypeptides, R¹ can denotehydrogen. On the other hand, R¹ can also denote an alkyl residue e.g.methyl or ethyl and in particular methyl since N-alkylation of the amidebond often has a major influence on the activity (cf. e.g.Levian-Teitelbaum et al., Biopolymers 28 (1989), 51-64).

[0033] The α-aminocarboxylic acids can also be used as monomericbuilding blocks in the form of L-enantiomers or/and D-enantiomers. Thespatial structure of the peptides according to the invention can bemodified by changing the chirality which can also influence theactivity. Retro-inverso peptides are particularly preferred i.e. peptides which are present in a reversed sequence direction and containD-amino acids as monomeric building blocks. In these retro-inversostructures the functional side chains have a similar spatial orientationto those in the native peptide sequence, but their biologicaldegradation can be impaired due to the presence of D-amino acids andthey therefore have advantages as drugs (cf. for example Wermuth et al.,J. Am. Chem. Soc. 119 (1997), 1328-1335 and references cited therein).

[0034] The peptides according to the invention ate preferably cycliccompounds in which in particular the monomeric building blocks X²¹ andX²⁹ are bridged together. This bridging can for example utilize the sidechains of the respective α-aminocarboxylic acid residues in which casebridging by means of disulfide bonds e.g. between two cysteine residuesis particularly preferred. Other types of cyclization between amino acidside chains are, however, also possible e.g. amide bonds between anamino acid with an amino side group e.g. ornithine or Lys and an aminoacid with a carboxylic acid side group such as Asp or Glu. In additionthe disulfide bridge can also be replaced by an alkylene bridge in orderto increase the chemical stability. In addition an amino acid side chainmay also be linked to the peptide backbone e.g. an ω-amino side groupmay be linked to the C-terminal end or a carboxylic acid side group maybe linked to the N-terminal end. A linkage of the N-terminus andC-terminus is also possible.

[0035] It is particularly preferred when at least one of the amino acidsX²¹, X²⁷, X²⁹ and X³⁰ is a D-amino acid. At least one of the amino acidsX²¹ to X³⁰ is particularly preferably a D-amino acid e.g. D-cysteine.

[0036] Instead of the disulfide bridge it is also possible to useso-called turn mimetics (Haubner et al., J. Am. Chem. Soc. 118 (1996),7884-7891) or sugar amino acids (Graf von Rödern et al., J. Am. Chem.Soc. 118 (1996), 10156-10167).

[0037] The peptides according to the invention can be obtained bychemical synthesis as elucidated in the examples. Alternatively thepeptides according to the invention can also be components ofrecombinant polypeptides.

[0038] Yet a further subject matter of the present invention arepeptides which are derived from the linear peptide uPA (19 to 31) andcyclic derivatives thereof and carry D-amino acid residues at selectedpositions. Such peptides have the general structural formula (II):

X¹—[X²]_(n)—[X³]_(m)—X⁴—K—Y—F—X⁵—X⁶—I—X⁷—W—[X⁸]_(r)  (II)

[0039] in which

[0040] X¹ to X⁸ each denotes an aminocarboxylic acid preferably anα-aminocarboxylic acid and X¹ and X⁷ or X¹ and X⁸ are optionally bridgedtogether,

[0041] n, m and r are each independently 0 or 1,

[0042] K is defined as X²³ and preferably denotes an α-amino-carboxylicacid with a lysine side chain,

[0043] Y is defined as X²⁴ and preferably denotes an α-amino-carboxylicacid with a tyrosine side chain,

[0044] F is defined as X²⁵ and preferably denotes an α-amino-carboxylicacid with a phenylalanine side chain,

[0045] I is defined as X²⁸ and preferably denotes an α-amino-carboxylicacid with an isoleucine side chain,

[0046] W is defined as X³⁰ and preferably denotes an α-amino-carboxylicacid with a tryptophan side chain

[0047] and the monomeric building blocks are linked by —CONR¹— or—NRLCO— bonds where R¹ in each case independently denotes hydrogen,methyl or ethyl and pharmaceutically compatible salts and derivativesthereof and in which at least one of the amino acid residues denotes X¹,X², X³, X⁶, I, X⁷, W and X⁸ denotes a D-amino acid residue.

[0048] The monomeric building blocks X¹ to X⁸ preferably have thefollowing meanings:

[0049] X¹ and—if present—X⁸ correspond to the meaning of X²¹ and X²⁹ andare e.g. α-aminocarboxylic acid building blocks with an SH side chain,in particular with a cysteine side chain.

[0050] X²—if present—is an α-aminocarboxylic acid with an aliphatic anduncharged side chain e.g. valine, leucine or isoleucine, in particularvaline.

[0051] X³ and X⁵ correspond to the meaning of X²⁶ and are e.g.α-aminocarboxylic acids with an aliphatic hydrophilic side chain such asserine or threonine, in particular serine.

[0052] X⁴ and X⁶ correspond to the meaning of X²² and X²⁷ and are e.g.α-aminocarboxylic acids with an aliphatic hydrophilic side chain, inparticular an amide side chain such as asparagine or glutamine, inparticular asparagine.

[0053] If not bridged with X¹, X⁷ is preferably a basicα-aminocarboxylic acid, in particular histidine. If it is bridged withX¹, then X⁷ is an α-aminocarboxylic acid with an SH side group, inparticular cysteine.

[0054] The present invention additionally concerns a pharmaceuticalcomposition which contains at least one peptide or polypeptide asdefined above as the active substance optionally together with commonpharmaceutical carriers, auxiliary agents or diluents. The peptides orpolypeptides according to the invention are used especially to produceuPA antagonists which are suitable for treating diseases associated withthe expression of UPAR especially for treating tumours.

[0055] An additional subject matter of the present invention is the useof peptides derived from the uPA sequence and in particular of uPAantagonists such as the above-mentioned peptides and polypeptides toproduce targeting vehicles e.g. liposomes, viral vectors etc. forUPAR-expressing cells. The targeting can be used for diagnosticapplications to steer the transport of marker groups e.g. radioactive ornon-radioactive marker groups. On the other hand the targeting can befor therapeutic applications e.g. to transport pharmaceutical agents andfor example also to transport nucleic acids for gene therapy.

[0056] The pharmaceutical compositions according to the invention can bepresent in any form, for example as tablets, as coated tablets or in theform of solutions or suspensions in aqueous or non-aqueous solvents. Thepeptides are preferably administered orally or parenterally in a liquidor solid form. When they are administered in a liquid form, water ispreferably used as the carrier medium which optionally containsstabilizers, solubilizers or/and buffers that are usually used forinjection solutions. Such additives are for example tartrate or boratebuffer, ethanol, dimethyl sulfoxide, complexing agents such as EDTA,polymers such as liquid polyethylene oxide etc.

[0057] If they are administered in a solid form, then solid carriersubstances can be used such as starch, lactose, mannitol, methylcellulose, talcum, highly dispersed silicon dioxide, high molecularfatty acids such as stearic acid, gelatin, agar, calcium phosphate,magnesium stearate, animal and vegetable fats or solid high molecularpolymers such as polyethylene glycols. The formulations can also containflavourings and sweeteners if desired for oral administration.

[0058] The therapeutic compositions according to the invention can alsobe present in the form of complexes e.g. with cyclodextrins such asγ-cyclodextrin.

[0059] The administered dose depends on the age, state of health andweight of the patient, on the type and severity of the disease, on thetype of treatment, the frequency of the administration and the type ofdesired effect. The daily dose of the active compound is usually 0.1 to50 mg/kilogramme body weight. Normally 0.5 to 40 and preferably 1.0 to20 mg/kg/day in one or several doses are adequate to achieve the desiredeffects.

[0060] The invention is further illustrated by the examples described inthe following and the figures.

[0061]FIG. 1 shows the quantity-dependent inhibition of the binding ofpro-uPA to a cell surface-associated uPAR by synthetic peptides;

[0062]FIG. 2 shows the competition of synthetic peptides with ATF forbinding to the uPAR;

[0063]FIG. 3A shows the structure of cyclo¹⁹⁻³¹ uPA 19-31 (right)compared to the structure of the corresponding domain from native uPAand

[0064]FIG. 3B shows the structure of the cyclic peptide derivativecyclo^(21,29) [Cys21,29]uPA₂₁₋₃₀.

[0065]FIG. 4 shows the inhibition of the uPA/uPAR interaction bysynthetic peptides and

[0066]FIG. 5 shows the inhibition of tumour growth in naked mice byadministration of synthetic peptides.

EXAMPLES

[0067] 1. Methods

[0068] 1.1 Solid Phase Peptide Synthesis

[0069] Linear peptides were synthesized on a 2-chlorotrityl resin(Barlos et al., Int. J. Pept. Protein Res. 37 (1991), 513 to 520) usingan Applied Biosystems Model 431 A peptide synthesizer or a multiplepeptide synthesizer model Syro II (MultiSynTech). Using the orthogonalFmoc strategy (Carpino and Han, J. Org. Chem. 37 (1972), 3404-3409;Fields and Noble, Int. J. Peptide Protein Res. 35 (1990), 161-214) theamino acid side chains were blocked with the protecting groups trityl(Asn, Cys, Gln and His), tert.-butyloxycarbonyl (Lys and Trp),tert.-butyl (Asp, Glu, Ser, Thr and Tyr), acetamidomethyl (Cys) and2,2,5,7,8-pentamethylchroman-6-sulfonyl or2,2,4,6,7-pentamethyldihydrobenzofuran-5-sulfonyl (Arg). The couplingwas carried out at room temperature in dimethylformamide using athree-fold excess of2-(1H-benzotriazol-1-yl)-1,1,3,3-tetramethyl-uroniumtetrafluoroborate/1-hydroxybenzotriazole/Fmoc-aminoacid with 2.5 equivalents of N-ethyldiiso-propylamine inN-methyl-pyrrolidone. The Fmoc group was removed by sequential treatmentof the resins with an excess of 40% or 20% piperidine indimethylformamide. The cleavage of the peptides and removal of the sidechain protecting groups was carried out simultaneously by treatment with82.5% trifluoroacetic acid/5% phenol/2.5% ethane dithiol/5%thioanisol/5% H₂O (0° C./1 h; room temperature/1 h). In the case of Arggroups protected with 2,2,5,5,7,8-pentamethylchroman-6-sulfonyl, thepeptides were incubated for an additional 12 h at room temperature. Thecrude peptides were precipitated at −30° C. with diethyl ether,dissolved in methanol, precipitated as previously described, dissolvedin tert.-butanol and lyophilized. Peptides containing tryptophan wereadditionally treated for 2 h with 5% acetic acid before thelyophilization.

[0070] The peptides were purified by HPLC using a reversed phase C18column (Nucleosil 1005-C18) or a YMC pack ODS column. They were cyclizedby forming a disulfide bridge between the cysteine residues. Theoxidation required for this was carried out by taking 0.1 to 0.3 mg/mlof the purified linear peptides up in 80% water and 20% DMSO (vol/vol)and removing the solvent under reduced pressure after 10 h. The cyclicpeptides were again purified by HPLC as described above.

[0071] 1.2 Mass Spectroscopy and Amino Acid Analysis

[0072] The purified and desalted peptides were analysed on a HPLC system140 B (Applied Biosystems, Foster City, USA). The UV absorbance wasmeasured with a UVIS 200 detector (Linear Instruments, Reno, USA) at 206nm. The chromatography was carried out on an Aquapore 3μ (AppliedBiosystems, Foster City, USA) reversed phase column (1 mm×50 mm) at aflow rate of 20 μl/min. The solvent system was 0.1% TFA in water (A) and0.1% TFA in acetonitrile (B). The HPLC system was coupled to anatmospheric pressure ionisation source which was connected to a tandemquadrupole instrument API III (Sciex, Perkin Elmer, Thornhill, Canada).

[0073] The quadrupole M/Z scale was calibrated with the ammoniumaddition products of polypropylene glycol. The average mass values werecalculated from the M/Z peaks in the charge distribution profiles of themultiple charged ions (Covey et al., Rapid Commun. Mass Spectrom. 2(1988), 249-256; Fenn et al., Science 246 (1989), 64-71).

[0074] The amino acid analysis was carried out according to theninhydrin method using the analytical system 6300 (Beckman Instruments,Fullerton, USA) after hydrolysing the peptides by the TFA-HCl vapourphase method which allows a quantitative determination of the peptideconcentration (Tsugita et al., J. Biochem. 102 (1987), 1593-1597).

[0075] 1.3 Flow Cytometry

[0076] The ability of the synthetic peptides to inhibit the uPA/uPARinteraction was determined by means of flow cytometry on a FACScan flowcytometer (Becton-Dickinson, Heidelberg, Germany) using the humanpromyeloid cell line U937 as a source of cellular native uPAR(Chuchulowski et al., Fibrinolysis 6, Suppl. 4 (1992), 95-102; Magdolenet al., (1996), supra). The U937 cells were stimulated with 1 mMphorbol-12-myristate-13-acetate (PMA) for 48 h. After stimulation withPMA the U937 cells expressed considerable amounts of cellsurface-associated uPAR.

[0077] The stimulated cells were treated for 1 min at room temperaturewith 50 mM glycine HCl, 0.1 NaCl, pH 3.6 in order to dissociateendogenous receptor-bound uPA. Subsequently the acidic buffer wasneutralized with 0.5 M HEPES-100 mM NaCl, pH 7.5. The cells were thenimmediately washed twice with PBS/0.1% bovine serum albumin (BSA) andcentrifuged for 10 min at room temperature and 300×g. The cells wereresuspended in PBS/0.1% BSA, adjusted to a concentration of 10⁶ cellsper ml and simultaneously incubated for 45 minutes at room temperaturewith 16 ng FITC-conjugated pro-uPA and various amounts of the syntheticpeptides. Before the analysis, propidium iodide, a fluorescent dye whichspecifically binds double-stranded DNA, was added to each sample inorder to determine the viability of the analysed U937 cells. Damaged,propidium iodide-labelled cells were excluded from the analysis.

[0078] 1.4 Solid Phase uPAR/uPA Binding Test

[0079] In addition to the flow cytometric analyses, a solid phaseATF-ligand binding test was carried out in order to examine theinteractions of synthetic peptides with the uPAR. For this microtitreplates were coated with recombinant human uPAR from CHO cells (Wilhelmet al., FEBS Lett. 337 (1994), 131-134; Magdolen et al., Electrophoresis16 (1995), 813-816) and the remaining protein-binding sites weresaturated with 2% BSA (weight/vol). After incubation with the samples(0.6 ng ATF together with 15 μg synthetic peptide per ml) and severalwash steps, the amount of ATF which had bound to the uPAR immobilized onthe microtitre plate was determined using a biotinylated monoclonalmouse antibody against the kringle domain of ATF (No. 377, AmericanDiagnostics, Greenwich, Conn., USA) and subsequent addition ofavidin-peroxidase conjugate and 3,3′, 5,5′-tetramethylbenzidine/H₂O as asubstrate for the peroxidase. The presence of synthetic peptides whichcompete with the ATF binding to UPAR reduces the conversion of thechromogenic substrate.

[0080] 2. Results

[0081] 2.1 Determination of the uPAR Binding Capacity of SyntheticPeptides by Quantitative Flow Cytometric Analysis

[0082] A comparison was made of the inhibitory capacity of the peptidesuPA₁₂₋₃₂ [C19A] (Appella et al., (1987), supra) the so-called clone20-peptide AEPMPHSLNFSQYLWYT (Goodson et al., (1994), supra) which wasidentified as the most effective peptide from a phage peptide libraryand of the synthetic peptide uPA₁₆₋₃₂ derived from the wild-type uPAsequence.

[0083] For this the purified peptides were analysed by massspectroscopy, quantified by amino acid analysis and then tested by flowcytometry according to the method described in 1.3 for their ability toinhibit the binding of fluorescent-labelled pro-uPA to the uPA receptoron U937 cells. It was found that pro-uPA is displaced in adose-dependent manner from the cell surface-associated uPAR by all threesynthetic peptides (FIG. 1). An approximately 15,000 to 12,000 molarexcess of uPA₁₂₋₃₂ [C19A] or clone 20 peptide resulted in a 50%inhibition of the binding of uPA. The peptide uPA₁₆₋₃₂ exhibited a 4- to5-fold higher affinity to uPAR compared to the two other peptides: anapproximately 3,000-fold molar excess is sufficient to achieve a 50%inhibition.

[0084] Furthermore it was found that the linear peptide uPA₁₉₋₃₁surprisingly has an IC50 value of ca. 0.8 μM whereas the IC50 value foruPA₁₆₋₃₂ is ca. 3.2 μM.

[0085] 2.2 Determination of the uPAR Binding Capacity of SyntheticPeptides in a Microtitre Plate Solid Phase Ligand binding Test

[0086] A series of peptides with variable sequence regions from thereceptor binding region of uPA were synthesized and were increasinglyshortened at the amino terminus starting with uPA₁₀₋₃₂. The microtitreplate solid phase binding test described in 1.4 was used to determinethe inhibitory capacity of these peptides. The results of this test areshown in FIG. 2.

[0087] It can be seen in FIG. 2A that the peptides uPA₁₀₋₃₂, uPA₁₂₋₃₁,uPA₁₄₋₃₂ and uPA₁₆₋₃₂ effectively inhibit the binding of ATF to uPAR.The peptides uPA₁₇₋₃₂ and uPA₁₈₋₃₄ have considerably reduced uPARbinding capacities. The peptide uPA₂₀₋₃₄ does not bind at all to theuPAR. In a further experiment the binding capacity of the peptidesuPA₁₉₋₃₁, uPA₁₈₋₃₀, uPA₂₀₋₃₂ and uPA₂₀₋₃₀ was tested. The result of thisexperiment is shown in FIG. 2B. Surprisingly it was found that uPA₁₉₋₃₁binds to the uPAR with higher affinity than the longer peptide uPA₁₆₋₃₂.The other tested linear peptides had no significant binding capacity.

[0088] The cyclic peptide cyclo¹⁹⁻³¹uPA₁₉₋₃₁ which contains anintramolecular disulfide bond between the cysteine residues at positions19 and 31 was surprisingly still able to inhibit the binding of uPA tothe uPA receptor. Furthermore the binding activity of cyclo¹⁹⁻³¹uPA₁₉₋₃₁was significantly more stable after long storage in aqueous solution orrepeated freeze/thaw cycles than that of the linear peptide uPA₁₉₋₃₁.

[0089] 2.3 Systematic Replacement of L-Amino Acids by D-Amino Acids inChemically Synthesized Linear and Cyclic Peptides From the RegionuPA₁₉₋₃₁

[0090] The uPAR binding capacity of synthetic linear and cyclic peptidesfrom the region uPA₁₉₋₃₁ was determined by in each case replacing oneL-amino acid by the corresponding D-amino acid. The results of thisexperiment are shown in the following table 1. TABLE 1 D-amino acidPeptide structure Inhibition Trp30 [D-Trp³⁰]uPA₁₉₋₃₁ ++ Trp30cyclo[D-Trp³⁰]uPA₁₉₋₃₁ + His29 [D-His²⁹]uPA₁₉₋₃₁ ++ His29cyclo[D-His²⁹]uPA₁₉₋₃₁ + Asn27 [D-Asn²⁷]uPA₁₉₋₃₁ ++ Asn27cyclo[D-Asn²⁷]uPA₁₉₋₃₁ ++ Ser21 [D-Ser²¹]uPA₁₉₋₃₁ ++ Ser21cyclo[D-Ser²¹]uPA₁₉₋₃₁ ++ Val20 [D-Val²⁰]uPA₁₉₋₃₁ ++ Val20cyclo[D-Val²⁰]uPA₁₉₋₃₁ + Cys19 [D-Cys¹⁹]uPA₁₉₋₃₁ +++ Cys19cyclo[D-Cys¹⁹]uPA₁₉₋₃₁ +++ cyclo19-31 cyclo[19-31]uPA₁₉₋₃₁ +++

[0091] It can be seen from this table that the introduction of D-aminoacids at positions Cys19, Val20, Ser21, Asn27, His29 and Trp30 in thelinear as well as in the cyclic peptides is possible without loss of theinhibitory effect. Moreover it was found that in the case of the linearpeptides the inhibitory effect is not lost by introducing D-amino acidsat positions Ile28 and Cys31.

[0092] 2.4 Synthesis of Modified Cyclic uPA Peptides

[0093] Using cyclo^(19,31)uPA₁₉₋₃₁ as the lead structure, a cyclicpeptide was prepared in which certain amino acids were deleted and/orsubstituted by other amino acids. The structure of this new syntheticpeptide variant cyclo^(21,29)[Cys21,29]uPA₂₁₋₃₀ is shown in FIG. 3. Incontrast to the synthesis method stated in 1.1 this peptide was preparedon a trityl chloride polystyrene resin.

[0094]FIG. 4 shows the inhibitory effect of this synthetic peptidevariant compared to cyclo^(19,31)uPA₁₉₋₃₁ andcyclo^(19,31)[D-Cys19]uPA₁₉₋₃₁.

[0095] 2.5 In Vivo Effect

[0096] 6×10⁶ human breast cancer cells MDA-MB-231 (Price et al., CancerRes. 50 (1990), 717-721) in a total volume of 300 μl were injected intothe right side of 4-6 week old Balbc/3 naked mice. Before injection thecancer cells were mixed with 200 μg of the cyclic UPA peptidescyclo^(19,31)uPA₁₉₋₃₁ and cyclo^(21,29)[Cys21]uPA₂₁₋₃₀ in PBS, pH 7.4.Subsequently the mice were treated twice weekly intraperitoneally withthe respective peptide at a dose of 10 mg/kg body weight (injectionvolume 300 μl). The volume of the primary tumours which occurred in themice in cm³ was determined after 1, 2, 3 and 5 weeks by measuring thetwo largest diameters. The control mice were administered PBS pH 7.4.Each group was composed of 5 mice. The results for the peptidecyclo^(19,31)uPA₁₉₋₃₁ are shown in Tab. 2. TABLE 2 Week Control uPApeptide 1 0 0 2 0.34 ± 0.3 0.086 ± 0.047 3 0.71 ± 0.5 0.303 ± 0.129 5 2.33 ± 0.32  0.62 ± 0.21*

[0097] The volume of the primary tumour after a five week treatment isshown in FIG. 5. It can be seen that the administration of both peptidesled to a significant reduction of the tumour growth in vivo.

1. A method of inhibiting the binding of at least one urokinaseplaminogen activator to at least one urokinase plaminogen activatorreceptor in a patient in need of such inhibition comprising:administering to said patient a peptide comprising monomeric buildingblocks and having the general structural formula (I):

(SEQ ID NO:4 in which X²¹ to X³⁰ each denotes an aminocarboxylic acidand X²¹ and X²⁹ are bridged together, Y is a spacer group that cancouple the peptide to carrier substances n and m are each independently0 or 1, and the monomeric building blocks are linked by —NR¹CO— or—CONR¹— bonds where R¹ in each case independently denotes hydrogen,methyl or ethyl, and wherein the amino acid residues X²¹-X³⁰ eachindependently have one of the following meanings: (i) X²¹ and X²⁹ areeach independently an aminocarboxylic acid residue with an SH side chainor X²¹ and X²⁹ are together two aminocarboxylic acid residues which arebridged by a thioether bond; (ii) X²² and X²⁷ are each independently anaminocarboxylic acid residue with an aliphatic side chain; (iii) X²³ isan aminocarboxylic acid residue with a basic or an aliphatic hydrophilicside chain; (iv) X²⁴, X²⁵ and X³⁰ are each independently anaminocarboxylic acid residue with an aromatic side chain, (v) X²⁶ is anaminocarboxylic acid residue with an aliphatic side chain, and (vi) X²⁸is an aminocarboxylic acid residue with an aliphatic side chain; and apharmaceutically compatible salt or derivative thereof, wherein saidderivative comprises a peptide of formula I in which reactive groups ofa side chain and/or of the N-terminus or C-terminus have been subjectedto one or more modifications, said modifications being selected from thegroup consisting of acylation, amidation and esterification ofcarboxylic acid groups.
 2. The method of claim 1, wherein X²¹ and X²⁹are bridged via a disulfide bond.
 3. A peptide comprising monomericbuilding blocks and having the general structural formula (II):X¹—[X²]_(n)—[X³]_(m)—X⁴—K—Y—F—X⁵—X⁶—I—X⁷—W—[X⁸]_(r)  (II) wherein, X¹,X², X³, X⁴, X⁵, X⁶, X⁷ and X⁸ are each independently an aminocarboxylicacid, n, m and r are each independently 0 or 1, K is anα-aminocarboxylic acid with a lysine side chain, F is anα-aminocarboxylic acid with a phenylalanine side chain, I is anα-aminocarboxylic acid with an isoleucine side chain, w isα-aminocarboxylic acid with a tryptophan side chain, and the monomericbuilding blocks are linked by —NR¹CO— or —CONR¹— bonds, wherein R¹ ineach case independently denotes hydrogen, methyl or ethyl, orpharmaceutically compatible salts and derivatives thereof, wherein atleast one of the amino acid residues X¹, X², X³, X⁶, I, X⁷, W and X⁸ isa D-amino acid residue.
 4. The peptide of claim 3, wherein the monomericbuilding blocks X¹ and X⁷, or X¹ and X⁸ are bridged together.
 5. Apharmaceutical composition comprising at least one peptide of claim 3and a pharmaceutical acceptable carrier thereof, and optionally at leastone auxiliary agent and/or diluent.
 6. A method of inhibiting thebinding of at least one urokinase plaminogen activator to at least oneurokinase plaminogen activator receptor in a patient in need of suchinhibition comprising: administering to said patient at least onepeptide of claim 3 in a binding of urokinase plaminogen activator tourokinase plaminogen activator receptor inhibting amount.
 7. The peptideor of claim 3, wherein X¹ and X⁸ are (a) each independently anaminocarboxylic acid residue with an SH side chain or (b) areaminocarboxylic acid residues which are adapted to be bridged by athioether bond.
 8. The peptide of claim 7, wherein X¹ and X⁸ are bridgedvia a disulfide bond formed between said SH side chains.
 9. The peptideof claim 7, wherein X¹ and X⁸ are bridged via a thioether bond.
 10. Thepeptide of claim 3, wherein X² is an aminocarboxylic acid residue withan aliphatic and uncharged side chain.
 11. The peptide of claim 10,wherein said chain is a valine, leucine or isoleucine side chain. 12.The peptide of claim 3, wherein X³ and X⁵ are each independently anaminocarboxylic acid residue with an aliphatic hydrophilic side chain.13. The peptide of claim 12, wherein said side chain is a serine orthreonine side chain.
 14. The peptide of claim 3, wherein X⁴ and X⁶ areeach independently an aminocarboxylic acid residue with an aliphatichydrophilic side chain.
 15. The peptide of claim 14, wherein said sidechain is an amide side chain.
 16. The peptide of claim 15, wherein saidside chain is an asparagine or glutamine side chain.
 17. The peptide ofclaim 3, wherein X¹ and X⁷ are each independently (a) a basicaminocarboxylic acid residue or (b) an aminocarboxylic acid residue witha SH side chain.
 18. The peptide of claim 17 wherein said basicaminocarboxylic acid residue is histidine.
 19. The peptide of claim 17wherein said side chain in (b) is a cysteine side chain.
 20. The peptideof claim 19 wherein X¹ and X⁷ are bridged via a disulfide bridge. 21.The method of claim 6, wherein said peptide is an antagonist ofurokinase plaminogen activator.
 22. The method of claim 6, wherein saidpatient suffers from a disease associated with the expression ofurokinase plaminogen activator receptor.
 23. The method of claim 22,wherein said disease is a tumor.
 24. The method of claim 6, wherein saidat least one peptide is administered to said patient via a targetingvehicle.
 25. A targeting vehicle comprising at least one peptide ofclaim
 3. 26. The targeting vehicle of claim 25, wherein said targetingvehicle is a viral vector or a liposome.
 27. The peptide of claim 2,wherein X²¹ and X²⁹ are bridged via a disulfide bond.
 28. The method ofclaim 1, wherein said peptide is a cyclic peptide and acts as anurokinase plaminogen activator antagonist.
 29. The method of claim 1,wherein said peptide is administered to said patient via a targetingvehicle.
 30. The method of claim 1, wherein said patients suffers from adisease associated with the expression of urokinase plaminogen activatorreceptor.
 31. The method of claim 30, wherein said disease is a tumor.32. The method of claim 31, wherein said peptide is part of (or iscoupled to) a polypeptide.
 33. (New) A targeting vehicle comprising apolypeptide comprising (or having coupled thereto) at least one peptidehaving the general structural formula (I):

(SEQ ID NO:4 in which X²¹ to X³⁰ each denotes an aminocarboxylic acidand X²¹ and X²⁹ are bridged together, Y is a spacer group that cancouple the peptide to carrier substances n and m are each independently0 or 1, and the monomeric building blocks are linked by —NR¹CO— or—CONR¹— bonds where R¹ in each case independently denotes hydrogen,methyl or ethyl, and wherein the amino acid residues X²¹—X³⁰ eachindependently have one of the following meanings: (i) X²¹ and X²⁹ areeach independently an aminocarboxylic acid residue with an SH side chainor X²¹ and X²⁹ are together two aminocarboxylic acid residues which arebridged by a thioether bond; (ii) X²² and X²⁷ are each independently anaminocarboxylic acid residue with an aliphatic side chain; (iii) X²³ isan aminocarboxylic acid residue with a basic or an aliphatic hydrophilicside chain; (iv) X²⁴, X²⁵ and X³⁰ are each independently anaminocarboxylic acid residue with an aromatic side chain, (v) X²⁶ is anaminocarboxylic acid residue with an aliphatic side chain, and (vi) X²⁸is an aminocarboxylic acid residue with an aliphatic side chain; and apharmaceutically compatible salt or derivative thereof, wherein saidderivative comprises a peptide of formula I in which reactive groups ofa side chain and/or of the N-terminus or C-terminus have been subjectedto one or more modifications, said modifications being selected from thegroup consisting of acylation, amidation and esterification ofcarboxylic acid groups.